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Publication numberUS3915949 A
Publication typeGrant
Publication dateOct 28, 1975
Filing dateFeb 12, 1974
Priority dateFeb 12, 1974
Also published asCA1062249A1, DE2505712A1
Publication numberUS 3915949 A, US 3915949A, US-A-3915949, US3915949 A, US3915949A
InventorsRobert L Colescott, Emil Kaiser, Charles D Bossinger, Paul I Cook
Original AssigneeArmour Pharma
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Solid phase synthesis of ACTH
US 3915949 A
Abstract
Resin peptides useful in the preparation of peptides having biological activity, and particularly such resin peptides containing R -CH2-Phe-Glu at one end of an amino acid chain, R being the resin and Phe and Glu being the residues of the amino acids phenylalanine and glutamic acid; and processes for the preparation of such resin peptides. Resin peptides are disclosed which contain amino acid chains identical with the amino acid chains of natural peptides having biological activity. Other resin peptides are disclosed which contain amino acid chains in which the amino acid residues differ in kind and sequence from amino acid chains of natural biologically active peptides but from which peptides having biological activity may be derived.
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Description  (OCR text may contain errors)

United States Patent [1 1 Colescott et al.

[ SOLID PHASE SYNTHESIS OF ACTH [75] Inventors: Robert L. Colescott, Bourbonnais;

Emil Kaiser, Chicago; Charles D. Bossinger, Olympia Fields; Paul 1. Cook, Kankakee, all of ill.

[73] Assignee: Armour Pharmaceutical Company,

Phoenix, Ariz.

22 Filed: Feb. 12, 1974 21 Appl. No.: 441,770

[52] US. Cl. 260/112.5; 424/179 [51] Int. Cl. C07C 103/52; A61K 37/40 [58] Field of Search 260/112.5, 78 A; 424/179 [56] References Cited OTHER PUBLICATIONS Yajima et al., Chem. Pharm. Bull., 18, 1333-1339, (1970).

Basjusz et al., Chem. Abstr., 711709362, (1969). Basjusz et al., Chem. Abstr., 71:77857h, (1969). Yamashiro et al., J. Am. Chem. Soc., 95, 1310-1315, (1973).

[ Oct. 28, 1975 Merrifield, Adv. in Enzymology, 32, 244-247, (1969).

Primary Examiner-Lewis Gotts Assistant Examiner-Reginald J. Suyat Attorney, Agent, or Firm-Frank T. Barber; Carl C.

Batz

[57] ABSTRACT Resin peptides useful in the preparation of peptides having biological activity, and particularly such resin peptides containing R -Cl-I Phe-G1u at one end of an amino acid chain, R being the resin and Phe and Glu being the residues of the amino acids phenylalanine and glutamic acid; and processes for the preparation of such resin peptides. Resin peptides are disclosed which contain amino acid chains identical with the amino acid chains of natural peptides having biological activity. Other resin peptides are disclosed which contain amino acid chains in which the amino acid residues differ in kind and sequence from amino acid chains of natural biologically active peptides but from which peptides having biological activity may be derived.

39 Claims, No Drawings soup PHASE SYNTHESIS or ACTH DESCRIPTION OF INVENTION Our total synthesis involves many reactions by which This invention relates to the synthesis of peptides and many new Intermediate resin peptIdes are formed and particularly resin peptides which are useful in the pro- I I duction of biologically active peptides. The invention 5 W6: proceed wlth the descnptlon Step by stepiglvmg involves Su,sh peptides as new compounds and also the structural formula, the general description and specesses by which they may be produced. Cific x mpl as W proceed.

In general, we utilize a solid phase synthesis in which BACKGROUND the insoluble polystyrene resin is chloromethylated. We It has long been know that certain natural iologi- 10 couple to the resin first phenylalanine, then glutamic cally active substances can be obtained from the glands acid and the other amino id f h h i i of animals and the substances so obtained utIlIzed m scribed sequence, using a system of protection and the tratment of deficiencies of the human body. One Protection of the active amine and carboxyl groups.

such substance is the adrenocorticotropic hormone, commonly called ACTH, which for many years has been obtained from the pituitary glands of animals, particularly porcine and bovine pituitary glands.

The burden of having to collectthe relatively small pituitary glands of animals at the time the animals are slaughtered, the limitation. to the quantity of such The hloromethylation 0f the resin is described by Following the coupling of the last amino acid in the chain the resin is cleaved from the peptide chain and the remaining protective groups removed.

Preparation of Insoluble Resin glands which can be collected and the extensive purifithe following structural formula:

cation procedures which are required to produce pep- Cl-CH,OCHu-Ch,-Cl CH 0H tides which can be administered to humans, are indeed in the above f l is the insoluble polystyrene formidable disadvantages to the preparation of natural resin Which is manufactured in head form by the cata Peptide hormones from fmimal a For many ye ars lytic polymerization of styrene and divinly benzene. the art has eagerly awaited dlscovery of pracmfal This resin is chloromethylated using chloromethyl methods and compounds which enable the commercial math I that and stannic chlorid cat I t synthesis of such peptides as ACTH from other than y e e ys animal Sources To our knowldege there have been no The reaction Is speclfically Illustrated by the followsuch compounds or methods prior to the discoveries of mg example the present invention. EXAMPLE 1 The human adrenocortIcotropIc hormone (ACTH) has been identified as having the following structure: one g 0f 2% nyl enzene rosslinked p0lystywhere the abbreviations Phe, Glu, Leu, etc. stand for rene resin 200-400 mesh was washed with three 2 liter the different amino acid groupings in th pep ide Ch portions of methylene chloride. Fine particles were reand the numbers represent the positions of the amino moved b d i i h methylene hl id ff h b acid g p in tha Chain according to accepted nomen' tom each time. The resin was washed with 2 liters of the clature. See the article by Riniker et al., in Nature New following solvents by suspension, stirring for 10 min- Biology, 114-115 (1972)- utes and filtration on a sintered glass Buchner: Two

lt is a principal object of this invention to discover intermediate resin peptides from which biologically active peptides may be derived, particularly peptides with adrenocortiocotropic hormone activity, and to provide effective processes for the commercial production of such peptides. Other more specific objects will become apparent as this specification proceeds.

We are aware of disclosures of certain laboratory methods for the synthesis of certain peptides of relatively short amino chain lengths. These include an artilowered to 00-500 with an ice-Water bathseventy-five cle by R. B. Merrifield entitled Solid Phase Peptide grams of anhydrous stannic chloride in 925 ml ice-cold Synthesis. 1. The Synthesis of a Tetrapeptide at pages chloromethyl methyl ether was added and the mixture 2149 to 2154 in Vol. 85 of Journal of the American stirred in the ice-bath for 2 hours. The resin was filtered Chemical Society (1963) and a book entitled Solid on a sintered glass Buchner and then washed with 2 Phase Peptide Synthesis" by John. W. Stewart and liter portions of the following solvents: 25% water in Janis D. Young published by'W. H. Freeman and Comdioxane, 25% two normal hydrochloric acid in dioxane, pany of San Francisco, California, but find in these water and twice with methanol. The washed resin was publications no disclosures of resin peptides having dried under vacuum at 4550C.. By this method the amino groups of the kind and in the sequence involved usual chloride content is between 0.7 to 1.0 milliin the present invention. equivalent per gram.

portions tetrahydrofuran, 2 portions water, 1 portion normal sodium hydroxide, 2 portions water, 2 portions dimethylformamide, 2 portions dioxane and 3 portions methanol. This washed resin was dried under vacuum at 60C.

Five hundred grams of this washed polystyrene resin was stirred with 5 liters of chloromethyl methyl ether at room temperature and then the temperature was PHENYLALAMINE ESTERIFICATION TO THE POLYSTYRENE RESIN By out synthesis phenylalanine is first bonded to the formula:

I &. cH,c| Hooc-cl cH,

NH I Coupling P Reaction No. l W H cH.-o c |CH BA.HCl

whereis polystyrene resin, BA is a suitable base such as triethylamine, diisopropylamine, diisopropylethylamine, or alkali metal salt, and P is an amino protective group which preferably is tertiary-butyloxycarbonyl (BOC) but may be amyloxycarbonyl (AMOC) or O- nitrophenylsulfenyl (NPS).

As illustrated by the above formula the tertbutyloxyl-phenylalamine is attached to the chloromethylated resin in the presence of an acid acceptor. This reaction is demonstrated by the following specific Example 2.

EXAMPLE 2 Fifty grams of chloromethylated polystyrene resin, prepared as above described, with chlorine content of 0.74 milli-equivalent (meq) per gram (37 meq chlorine) and 19.6 grams BOC-l-phenylalanine (74 meq) stirred in 150 ml of absolute ethyl alcohol and then 9.77 ml of triethylamine (72 meq) was added and the mixture refluxed with stirring for 24 hours. The mixture H polystyrene resin. This is described by the following 5 DEPROTECTION 1. Acid CH,O-CCNH, 2. Base This resulting resin peptide is designated Compound No. l.

The deprotection of the amine function of the phen- EXAMPLE 3 A 25 gram sample of the BOC-phenylalanine resin as prepared by Example 2 was placed in the reaction vessel of a peptide synthesizer. The sample was washed twice with 125 ml portions of methylene chloride for two minutes each. 125 ml of trifluroacetic acid in methylene chloride was added and the mixture reacted for 30 minutes. After filtration the resin was washed with three 125 ml portions of methylene chloride, 2 portions of methanol and 3 portions of chloroform, each was being of 2 minute duration. Neutralization was accomplished bya 5 minute reaction with 125 by a of a 10% solution of triethylamine and chloroform. The resin was then washed 3 times with 125 ml of chloroform and 3 times with 125 ml of methylene chloride.

COUPLING OF GLUTAMIC ACID was cooled, filtered on a sintered glass Buchner and washed on the Buchner with 500 ml portions of the following solvents: 2 times with 3A denatured alcohol, 2 times with dioxane, 2 times with 3A denatured alcohol, 2 times with water, 2 times with methanol. The resin was dried under vacuum at 4045C.. Nitrogen analysis will show values varying from about 0.50 to 0.70 meq per gram. When the BOC protecting group was removed with trifluoroacetic acid as hereinafter described and the resin titrated to determine the available terminal amine group, this sample was found to approximate 0.38 meq per gram.

In the above formula CA is a coupling agent which preferably is dicyclohexylcarbodiimide (DCC) but may be any coupling agent which forms peptide bonds, such as diimides, azides and active esters. The term B2 is benzyl or a benzyl derivative such as, for example, pmethoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, or benzhydryl. The symbols, BA, P, CA and B2 are to be taken as having the meanings above defined wherever they appear in this specification and claims.

Since the formula as above given begins to be cumbersome we may rewrite the formula of the reaction product in the following manner:

where Phe stands for the phenylalanine residue, Glu stands for the glutamic acid residue and P and B2 are as previously defined. This simplified nomenclature will be utilized in the description of all subsequent reactions.

In carrying out the reaction of the P, B2 and glutamic acid may be combined as in BOC-l-y-benzylglutamate and this added to the deprotected phenylalanine resin and the coupling promoted by addition of DCC. This coupling is then followed by deprotection just as explained in connection with the phenylalanie resin peptide. The resulting product following deprotection bears the formula:

-PheGluNH, (Compound No. 2)

We believe that this resin peptide was made for the first time by our invention, and that this is an important link in the synthesis of the hormone, ACTH.

Further we believe it is important that the coupling reaction be complete and have found the Ninhydrin test described by E. Kaiser, R. Colescott, C. Bossinger and P. Cook, the inventors herein, in Anal. Biochem. 34, 595-98 (1970) to be applicable to determine when the coupling reaction is sufficiently complete. If the Ninhydrin test is negative we may proceed to the deprotection of the resin peptide and go on to the following coupling reaction. If this test is positive we repeat the coupling step until the Ninhydrin test result is finally negative.

Following are specific examples of the coupling of glutamic acid:

EXAMPLE 4 To the deprotected phenylalanine resin with 10.7 meq of amine group was added a solution of millimoles (approx. 50% excess) of BOC-l--ybenzylglutamate in 100 ml of methylene chloride. After 2 minutes a solution of 15 meq of dicyclohexylcarbodiimide (DCC) was added and the mixture agitated for 45 minutes. The product was filtered and washed twice each with 125 ml portions of chloroform and methylene chloride. The Ninhydrin test was performed on a 3-5 mg sample of resin peptide reaction product and found to be negative. This resin was then deprotected as was described in Example 3.

EXAMPLE 4A Two grams of phenylalanine resin was deprotected and neutralized as previously described. Three millimoles of NPS-l-y'benzyl glutamate, dissolved in ml of methylene chloride, was added followed by 3 millimoles of dicyclohexylcarbodiimide. The mixture was agitated for one hour, filtered and washed with two portions of methylene chloride, two portions of methanol and three portions of methylene chloride.

EXAMPLE 4B In place of NPS in Example 4A we may substitute AMOC in the same meq amounts and substantially the same results may be expected.

EXAMPLE 4C In place of BOC-l-y-benzylglutamate we may use 15 millimoles of BOC-l-y-p-bromobenzylglutamate, and the reaction carried through in the manner set forth in Example 4. In this case we obtain the product in which the B2 group is p-bromobenzyl. After deprotection and neutralization we obtain Compound 2 the same as obtained in Example 4.

EXAMPLE 4D In Example 4 the Bz group is benzyl. Either pmethoxybenzyl, p-chlorobenzyl, p-nitrobenzyl or benzylhydrol may be substituted for the benzyl group in BOC-l-y-benzylglutamate, and the procedure carried out as set forth in Example 4 to obtain a reaction product in which the B2 group is p-methoxybenzyl, pchlorobenzyl, p-nitrobenzyl, or benzhydryl. After deprotection and neutralization we obtain in each case Compound 2, the same as obtained in Example 4.

The following Table 1 lists in sequence the amino acids attached at each of reactions 2 to 39 indicating the position in chain in which the attachment is made and listing the reactant used with the preferred protecting groups.

Table I Amino Acid Group Reaction Position Amino Acid With Preferred Number Number Being Attached Protectants 2 38 glutamic acid BOC-l-F-benzyl glutamate 3 37 leucine BOC-l-leucine hydrate 4 36 proline BOC-l-proline 5 35 phenylalanine BOC-l-phenylalanine 6 34 alanine BOC l-alanine 7 33 glutamic acid BOC-l-F-benzyl glutamate 8 32 alanine BOC-l-alanine 9 3l serine BOC-O-benzyl-lserine I0 30 glutamic acid BOC-l-Fbenzyl glutamate l l 29 aspartic acid BOC-l-B-benzylaspartate I2 28 glutamic acid BOC-l-F-benzyl glutamate I3 27 alanine BOC-l-alanine I4 26 glycine BOG-glycine I5 25 asparaginc BOC-l-asparagine-pnitrophenylester I6 24 proline BOC-l-proline I7 23 tyrosine BOC-l-tyrosinc (20% dimethylformamide for solubility) I8 22 valine BOC-l-valine I9 21 lysine BOC-2-chlorocarbobenzyloxy-l-lysine (10% dimethylformamide for solubility) 20 2O valine BOC-l-valine 21 19 proline BOC-l'proline 22 18 arginine BOC-l-tosylarginine (20% dimethylformamide for solubility) 23 17 arginine BOC-l-tosylarginine (20% dimethylformamicle for solubility) 24 I6 lysine BOC 2chlorocarbo- Amino Acid Group Reaction Position Amino Acid With Preferred Number Number Being Attached Protectants benzyloxy-l-lysine (10% dimethylformamide for solubility 25 I lysine BOC-2-chlorocarb0- benzyloxy-l-lysine dimethylformamide for solubility 26 I4 glycine BOC-glycine 27 I3 valine BOC-l-valine 28 12 proline BOC-l-proline 29 l l lysine BOC-Lchlorocarbobenzyloxy-l-lysine (10% dimethylformamide for solubility) 30 10 glycine BOC-glycine 3 l 9 tryptophan BOC-l-tryptophan (5% dimethylformamide for solubility) 32 8 arginine BOC-l-tosylarginine (20% dimethylformamide for solubility) 33 7 phenylalanine BOCJ-phenylalanine 34 6 histidine BOC-im-carbobenzyloxyl-histidine 35 5 glutamic acid BOC-l-F-benzylglutamate 36 4 methionine BOC-l-methionine 37 3 serine BOC-O-benzyl-lserine 38 2 tyrosine BOC-l-tyrosine 20% dimethylformamide for solubility) 39 l serine BOC-O-benzyl-lserine As was described in connection with the attachment of glutamic acid in reaction No. 2, each succeeding reaction to attach another amino acid group involves the same procedure in which the resin peptide previously prepared is coupled with another amino acid group under condition of protection, then the coupled peptide is deprotected and neutralized. More specifically, the following steps may in the case of each reaction be as follows:

Coupling:

l5 millimoles of the appropirate BOC-amino acid (0.43 equivalent excess in 100 ml of methylene chloride) millimoles of dicyclohexylcarbodiimide (coupling agent) in 15 ml of methylene chloride 40 minutes reaction time 2X 125 ml chloroform washes 2 minutes each 2X 125 ml methylene chloride 2 minutes each Deprotection:

2X 125 ml methylene chloride washes 2 minutes each 50% trifluoroacetic acid in methylene chloride 5 minutes 125 ml 50% trifluoroacetic acid in methylene chloride 25 minutes 125 ml 3X 125 ml methylene chloride washes 2 minutes each 2X 125 ml methanol washes 2 minutes each 3X 125 ml chloroform washes 2 minutes each Neutralization:

2X 125 ml 10% triethylamine in chloroform 5 minutes each 4X 125 ml chloroform washes 2 minutes each The procedures for making the coupling, the deprotection and neutralization steps in each of reactions 3 to 39 may be the same as already described in connection with reaction No. 2 except for the variances set forth in the following description.

As previously stated the Compound No. 2 which is the result of reaction N0. 2 (after deprotection and neutralization) is:

and Compound No. 3, which is the result of reaction No. 3, is:

and Compound No. 4, the result of reaction No. 4, is:

and, Compound No. 5, the result of reaction No. 5, is:

This pattern continues until the attachment of Asn at the 25th position. At this position the coupling agent DCC cannot be used because of a side reaction that destroys some of the asparagine so that this position the amino acid is coupled as an active ester.

The deprotected resin peptide is agitated with an active ester of asparagine such as p-nitrophenyl ester, ortho-nitrophenyl ester or penta-chlorophenyl ester.

This coupling is demonstrated more specifically by the following Example 5.

EXAMPLE 5 The resin peptide represented by Compound No. 14 obtained as a result of reaction No. 14 (after deprotection and neutralization) was washed with three portions of dimethylformamide for 2 minutes each. Three millimoles of BOC-l-asparagine-p-nitrophenyl ester dissolved in 15 ml of dimethylformamide was shaken with the resin for 16 hours, then washed with three portions of dimethylformamide, three portions of methanol and three portions of methylene chloride.

EXAMPLE 5A In place of the p-nitrophenyl ester of Example 5 either ortho-nitrophenyl ester or penta-chlorophenyl ester may be substituted, and the reaction carried out as set forth in Example 5 to accomplish the coupling of asparagine.

The coupling at position 25, by the active ester procedure is followed by the usual deprotection and neutralization and this results in the resin peptide compound No. 15 and is represented by the following formula:

In reactions Nos. 17 and 38, at positions Nos. 23 and of this specification we employ the symbol T to mean 2 respectively, where tyrosine is attached we prefer to tosyl or nitro. We also use the T protection group in use no protection on the phenolic hydroxyl of the tyrojoining arginine in reaction 23 at position 17, and in resine 'but may use a benzyl or benzyl derivative protec- 10 action 32 at position 8. tor. The symbol Y is defined to mean no protection or To illustrate, the structural formula for Compound benzyl or benzyl derivative. I No. 22 formed as a result of reaction No. 22, is given To illustrate, the formula for Compound No. 17 as follows:

formed as a result of reaction No. 17, we give the fol- For the coupling of the histidine in reaction No. 34 lowing structural formula: at position 6, we prefer to use as the imidazole protec- In reaction No. 19, position No. 21, where lysine is tion agent the carbobenzyloxy group (CBZ) but may attached, we prefer to use as the epsilon amine protecuse a tosyl, dinitrophenyl, benzyl, benzyl derivative or tion agent 2-chlorocarbobenzyloxy (Cl-CBZ) but may 35 no protecting group. We use the symbol W to indicate also use carbobenzyl (CBZ), bromocarbobenzyloxy, either no protective group or any of the above named 2,4-dichlorocarbobenzyloxy, or trifluoroacetyl (TFA). derivatives.

We use the symbol V to indicate that the epsilon pro- The symbols T, Y, V and W have the meanings as tection agent is one of these named groups. This V proabove defined whereever they appear throughout this tection agent is used also for attachment of lysine in 40 specification and claims. each of the reactions Nos. 24, 25 and 29 at positions To illustrate, the structural formula for Compound Nos. l6, l5 and 11 respectively. To illustrate, the struc- No. 34, formed as a result of reaction No. 34, is given tural formula for Compound No. 19 formed as a result as follows:

Ala-Gly-Asn-Pro-Tyr-Val-Lys-Val-Pro-Arg-Arg-Lys-Lys-Gly- T T V V of reaction No. 19 is given as follows: 55 Upon this attachment of serine in reaction 39 at the @-CH -Phe-(TluLeu--ProPhe-Ala-GluAla-?er(flu-Aspiilu B2 B2 B2 B2 B2 B2 For the coupling of the arginine amino acid in reacnumber one position, according to the manner and setion No. 22 at positions 18 we prefer to use as the quence above described,and the deprotection and neuguanidino protection agent the tosyl group (p-toluene tralization of the coupled resin peptide, we arrive at sulfonyl) but may use a nitro group, and in the formula Compound No. 39 which has the following formula:

l l in After each coupling reaction, and before deprotecl EXAMPLE 6 tion of the resin peptide we prefer to apply'the Ninhy- Two grams of compound 39 were placed in a drin test, and we find the test is not always negative revessel with 2 mls of anisole and lo mls of anhydrous quiring repeat of the coupling reaction last drogen fluoride was added by distillation. This mixture formedwas stirred at 0C for 1 hour. The hydrogen fluoride AS an example we found that when 25 grams of was removed by vacuum distillation, the residue BOG-phenylalanine resin was subjected to reactions 2 washed four times with ethyl acetate followed by to 39 of Table I the resulting Compound 39 (aftar traction with glacial acetic acid. The acetic acid extract deprotection, neutralization and drying under vacuum) was lyophilized to give 86 grams of a fluffy white weighed grams powder. This process removes the peptide from the After the Synthesis of the resin peptide and the resin and removes all protective groups on the amino tachment of all the desired amino acids in the desired acid sequence, this resin peptide, after the usual deprotec- The following Example 7 gives a Specific demonstra tion and neutralization steps, may be treated to remove tion of the cleavage reaction above referred to where the resin and the remaining protective groups. Suitably, v is TFA' the resin and most or all of the remaining protective EXAMPLE 7 groups may be removed by treatment with hydrogen fluoride. The formula for this cleavage reaction is: Two grams of the above blocked ACTH resin peptide CH PheGluLeu-Pro-PheAla-Glu-AlaSer-GluAsp-Glu-Ala- Bz Bz B2 B2 Bz Bz GlyAsnPro-Tyr-Val-LysValPro17g7rgZysLysGly-Val Y v T T J ProLyisGlyTrpArgPhe-HT-Glu-Met-Ser-Tfir-Se NH V T W Bz z Y Bz where V is other than TFA were placed in a Kel-F vessel with 2 ml of anisole and HF l0 mls of anhydrous hydrogen fluoride was added by l 40 distillation. This mixture was stirred at 0C for 1 hour. Reaction 40 The hydrogen fluoride was removed by vacuum distilla- SerTyr-SerMet-GluHis-PheArgTrp-GlyLys-Pro-Val-Gly-Lysl23456789l0lll2l3l4l5 --Lys-ArgArg-ProValLys-Val-Tyr-Pro-AsnGlyAlaGluAsp-Glu SerAla-Glu-AlaPheProLeuGlu-Phe which we call Compound 40. tion, the residue washed four times with ethyl acetate Where V in the above reaction is TFA the reaction followed by extraction with glacial acetic acid. The product is: acetic acid extract was lyophilized to give 856 mg of TFA Ser--Tyr-SerMetGluHisPheArg-Trp-GlyLysPro-Val-Gly 123 456789l0lll213l4 TFA TTA TFA LysLysArg-ArgProValLys-Val-TyrPro-AsnGlyAla-Glu l5 l6 l7 18 19 20 21 22 23 24 25 26 27 2s AspGluSer-AlaGluAla-PheProLeuGlu-Phe 29 30 31 32 33 34 35 36 37 38 39 (Compound 41) The following Example 6 gives a specific demonstrafluffy white powder. This process removes the peptide tion of the cleavage reaction above referred to where from the resin and removes all blocking groups on the V is other than TFA. difunctional amino acids except the trifluoroacetyl blocking group of-the lysine residues. Hence, this product is called TFA-ACTH peptide.

The TFA-ACTH peptide was treated with aqueous ammonium hydroxide for removal of the trifluoroacetyl groups from the lysine residue. Three hundred eighty mg of the TFA-ACTH was stirred with 100 ml of 4 normal ammonium hydroxide containing 0.1% mercaptoethanol for 16 hours. This generates the crude ACTH which had an ACTH activity of 40 units per mg when assayed by the U.S.P. method.

We have further discovered a synthesis of an active ACTH hormone of a sequence described by T H. Lee, A. B. Lerner and V. Buettner Janusch, J. Biol. Chem., 236,2970 (1961) (glutamine sequence) which we believe to be an improvement of the asparagine sequence ACTH hormone the synthesis of which has above been described. This peptide is easier to purify and is more alkali stable than the peptide having the asparagine sequence. This synthesis involves coupling of different amino acids at certain coupling reactions. These differences will become more apparent on reference to the following Table II which describes the coupling reactions of the synthesis of the glutamine sequence, when using preferred protecting groups.

TABLE II Amino Acid Group Reaction Position Amino Acid with Preferred Number Number Being Attached Protectants 30 glutamine BOC-l-glutamine-pnitrophenylester l l 29 aspartic acid BOC-l-B-bcnzyl aspartate 12 28 glutamic acid BOC-l-F-benzyl glutamate 13 27 glycine BOC-glycine 14 26 alanine BOC-l-alanine 15 2S aspartic acid BOC-l-fi-benzyl aspartate l6 24 proline BOC-l-proline 17 23 tyrosine BOCltyrosine dimethylformamide for solubility) l 8 22 valine BOC-l-valine 19 21 lysine BOC-e-trifluoroacetyl-l-lysine (5% dimethylformamide for solubility 20 20 valine BOC-l-valine 21 I9 proline BOC-lproline 22 18 arginine BOC-l-tosylargi nine (20% dimethylformamide for solubility) 23 17 arginine BOC-l-tosylarginine (20% dimethylformamide for solubility) 24 16 lysine BOC-e-trifluoracetyl -l-lysine(5% dimethylformamide for solubility) 25 15 lysine BOC-e-trifluoroacetyl -l-lysine (5% dimethylformamide for solubility) 26 14 glycine BOC-glycine 27 13 valine BOC-l-valine 28 I2 proline BOC-l-proline 29 l l lysine BOC-e-trifluoracetyl-l-lysine (5% dimethylforma' mide for solubility) 30 10 glycine BOC-glycine 3 l 9 tryptophan BOC-l-tryptophan 32 8 arginine BOC-l-tosylarginine (20% dimethylformamide for solubility) TABLE ll-Continued Amino Acid Group serine Reactions 2 to 9 at positions 39 to 3] are precisely the same as given in Table No. 1.

As seen from the above Table ll, our improved synthesis differs in that different amino groups are attached at the 30th, the 27th, the 26th and the 25th positions in the amino acid chain. Compounds 1 to 9 of the synthesis are the same as compounds 1 to 9 of the synthesis first described, but in reaction No. 10, where Gln is substituted for Glu the resulting compound which we wll call 10A has the formula:

in the reaction No. 13, where Gly is substituted for Ala, the resulting compound, which we call Compound 13A, has the formula:

in reaction No. 14, where Ala is substituted for Gly, the resulting resin peptide, which we call compound 14A, has the following formula:

in reaction No. 15, where Asp is substituted for Asn, the resulting resin peptide, which we call compound 15A, has the following formula:

,and the final coupling reaction No. 39 results in a resin 3X 150 ml methylene chloride 2 min. each peptide which we call Compound 39A, having the fol- BX 150 ml methanol 2 min. each lowing formula: 7 2X 150 ml trichloroethylene 2 min. each 82 Y V T T V V 1 B2 B2 Y 82 When Compound 39A (where V is TFA) is subjected Deprotection: to treatment with HF, it becomes: 15 2X 150 ml trichloroethylene washes 2 min.

TATFA TA Asp-GlnSer-Ala-Glu-Ala-PheProLeu-GluPhe (Compound 41A) and when treated with a suitable aqueous base such as each piperidine or ammonium hydroxide, to remove the 75 ml phenol in 4N-HCI dioxane +75 ml 50% TFA groups, becomes: 30 trichloroacetic acid in trichloroethylene with 4% Lys-Arg-Arg-Pro-Val-Lys--Val-Tyr-ProAspAlaGly-Cvlu-AspGln l6 l7 l8 19 20 21 22 23 24 25 26 27 28 29 30 SerAla-Glu-AlaPhe-Pro-LeuGluPhe (Compound 42) In cases where V is other than TFA, and when the mercaptoethanol for 30 min. Compound 39A is subjected to treatment with HF this 40 2X 150 ml chloroform 2 min. each compound becomes the Compound 42, with the struc- 2X 150 ml methanol 2 min. each ture above given. Compound 42 may be purified as de- 3X 150 ml chloroform 2 min. each scribed in connection with Compound 40, and is found Neutralization: to be biologically active as an ACTH hormone. 1x 150 ml 10% triethylamine in chloroform l0 5 min. EXAMPLE 8 4X 150 ml chloroform 2 min. each In this spe i ic e a p We fecofd'lhe synthesis on a The amino acids were coupled in the order set forth medium Seale, of Compound 42, and its Purification in Table II, and using the same combined amino acid obtain a biologically effective ACTH hormone prodd protecting groups as t f rth in T bl II, uct. In reaction 10 at position 30 the resin was washed Twentyfive grams of BOG-Phenylalanine resin P three times with dimethylformamide and after the neu- Pared as reaction 1, Previously described, was tralization step, 25 millimoles of BOC-l-glutamine-p- Placed in the reaction Vessel of a P p syrltheSizer nitrophenylester dissolved in 150 ml of dimethylformmarketed y SehWaYZ-Menn, of Orangeburg, New amide containing 1% acetic acid, was added and al- York. This apparatus ia for the automated synthesis o lowed to react for 16 hours. The resin was then washed P p y a p reading programmer- The resin used with three portions of dimethylformamide, 2 minutes had a 0397 meq/gm equivalency and 9-927 total each, with two portions of methanol, 2 minutes each, equivalents. The system of coupling, deprotection and d i h th trichloroethylene, 2 minute each. neturalization w r as fo l s! The resin was subjected to the Ninhydrin Test after Coupling: each coupling and this gave a positive result at the reac- 25 millimoles of ppr p amino acid 5 equivation 17 but gave a negative result after this reaction was lent excess) dissolved in 25 ml dimethylformamide, repeated. Upon completion of the 39 coupling ream- 25 ml anisole, 25 grams urethane and 75 ml of tions and after final deprotection and neutralization methylene chloride 10 min. stirring and drying under vacuum the yield was 40.5 grams. 25 millimoles of dicyclohexylcarbodiimide in ml Two grams of the protected resin peptide was reacted of trichloroethylene reaction time with stirring with hydrogen fluoride and anisole as previously defor 45 min. scribed. The yield was 822 mg of the peptide having TFA protecting groups still attached. A similar 4 gram cleavage gave 1,685'mg.

To purify the peptide compound, 1.35 grams of the peptide separate from the resin but still retaining its TFA groups was stirred with 135 ml of 0.2 molar piperidine containing 0.1% mercaptoethanol for 2 hours. This mixture was lypholized to give an off-white solid. This mixture was dissolved in 100 ml water and adjusted to pH 4.5 with acetic acid and absorbed on a carboxymethyl cellulose column of 750 ml bed volume. The impurities were eluted with 18 bed volumes of 8 mmho ammonium acetate buffer at pH 6.7. The active ACTH peak was then eluted with 1 1.5 mmho buffer at pH 6.7. The active fractions were lyophilized and desalted on a column of G-25 fine Sephadex. The lyophilized white fluffy powder yield was 361 mg. It had an ACTH activity of 92:11 units per milligram and had the correct amino acid composition.

EXAMPLE 9 In this specific example we record the synthesis, on a large scale, of compound 41A and its purification to obtain a biologically effective ACTH hormone prodnet.

1 16.5 grams of BOC-phenylalanine resin was placed in a specially built reactor for Schwarz-Mann Peptide Synthesizer. This resin had a titer of 0.57 meq/gm or 66.4 meq total. In this synthesis the coupling, deprotection, and neutralization steps were as follows: Coupling:

133 millimoles of appropriate BOC amino acid (1 equivalent excess) dissolved in 600 ml of methylene chloride 10 min. stirring 133 millimoles of dicyclohexylcarbodiimide in 133 ml of methlene chloride 45 min. reaction time 2X 750 ml methylene chloride 2 min. each 2X 750 ml methanol 2 min. each 3X 750 ml methylene chloride 2 min. each Deprotection:

2X 750 ml methylene chloride 2 min. each 50-50 mixture of trifluoroacetic acid and methylene chloride 30 min. 750 ml 3X 750 ml methlene chloride 2 min. each 3X 750 ml methanol 2 min. each Neutralization:

2X 750 ml 10% triethylamine in chloroform 5 min. each 2X 750 ml chloroform 2 min. each 2X 750 ml methlene chloride 2 min. each The sequence of the amino acids, the combined amino groups and protective groups and the proce dures followed were the same as in Example 8.

The Ninhydrin Test was applied at each coupling reaction and found to be positive at each of reactions Nos. 2, 22 and 23, but in each case the test showed negative after repeating the coupling step.

In reaction No. at position 30, the coupling involved the use of the p-nitrophenyl active ester as in Example 8.

The yield of the resin peptide (Compound No. 39A) after the final, or 39th, coupling and after deprotection, neutralization and drying under vacuum was 316 grams.

Cleavage:

100 grams of the above resin peptide was placed in a large scale Kel-F vessel with 5 grams of dithioerythritol and 100 ml of anisole. 350 ml of anhydrous hydrogen fluoride was added by distillation and the mixture stirred at 0C for 20 min. Then the hydrogen fluoride was removed by vacuum distillation. The residue was washed four times with 1 liter portions of ethyl acetate followed by extraction with glacial acetic acid. The acetic acid extract was lyophilized to give 47.6 grams of a fluffy white powder which is Compound 41A still retaining the TFA groups.

47.6 grams of the TFA peptide (Compound 41A) was stirred for 3 hours in 5.5 liters of 0.2 molar piperidine and one molar urea containing 0.1% mercaptoethanol. This solution was then adjusted to pH 4.0 with acetic acid and filtered through at 0.22 micron millipore filter. This solution was then ready for purification.

Purification The crude ACTH peptide, with the resin and all protecting groups removed, was absorbed on a column of carboxymethyl cellulose with a bed volume of 1800 ml. The impurities were eluted with 44 liters of ammonium acetate buffer at pH 6.7 and 4.0 mmhos. The ACTH peak was eluted with ammonium acetate buffer at pH 7.5 and 4.0 mmhos and lyophilized to a white powder. This powder was dissolved in 400 ml of 0.5 molar acetic acid containing 0.1% mercaptoethanol and desalted on a column of 16 liters bed volume of Sephadex G-25 superfine. The peptide containing peak was lyophilized to give 7.19 grams of fluffy white powder with the correct amino acid composition and an ACTH activity of 82:7 units per milligram.

While only certain embodiments of our invention have been described in specific detail it will be apparent to those skilled in this art that other specific embodiments may be practiced, and many changes made all within the spirit of the invention, and it is intended that all such other embodiments and changes be considered within the scope of the appended claims.

We claim:

1. A peptide having the structure:

T A TFA Ser-TynSer-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys- 123456789l01ll213l4l5 TFA TFA -Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu- 16 l7 I8 19 20 21 22 23 24 25 26 27 28 29 30 -Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe 2. A peptide having the structure:

TFA T A Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys'Pro-Val-Gly-Lys- 1234567891011l2l3l4l5 T A T A -Lys-Arg-Arg-Pro-VaI-Lys-Val-Tyr-Pro-Asp-Ala-Gly-Glu-Asp-Glnl6 l7 18 19 20 21 22 23 24 25 26 27 28 29 30 -Ser-AIa-Glu-Ala-Phe-Pro-Leu-Glu-Phe 3. A resin peptide having the structure:

and is divinylbenzene crosslinked polystyrene in which resin.

B2 is benzyl, p-methoxybenzyl, p-chlorobenzyl, p- 8. A resin peptide having the structure:

nitrobenzyl or benzylhydryl and is divinylbenzene crosslinked polystyrene 5 NH -Gln-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-CH,-

resin.

4. A resin peptide having the structure: B2 B2 32 NH -ProLeuGluPheCH in whlch 2 B2 is benzyl, p-methoxybenzyl, p-chlorobenzyl, p-

51 nitrobenzyl or benzylhydryl and is divinylbenzene crosslinked polystyrene in which resin 82 is benzyl, p-methoxybenzyl, p-chlorobenzyl, p- A resin peptide having the structure;

nitrobenzyl or benzylhydryl and is divinylbenzene crosslinked polystyrene resin. NH -Lys-Val-Tyr-Pro-Asp-Ala-Gly-Glu-Asp-Gln-Ser-Ala-Glu-Ala- 5. A resin peptide having the structure: v Y 82 B2 B2 B2 B2 Phe-Pro-Leu-Glu-Phe-CH, NH Glu-Se -Ala-Gl -Ala-Phe-Pro-Leu-GlIT-Phe-CH,-

2 B2 B2 B2 in which whl'ch B2 is benzyl, p-methoxybenzyl, p-chlorobenzyl, p-

82 is benzyl, p-methoxybenzyl, p-chlorobenzyl, pnitrobenzyl or benzylhydryl,

mtrobenzyl benzylhydryl is divinylbenzene crosslinked polystyrene resin, and is divinylbenzene crosslmked polystyrene Y is B2 or H and V is 2-chlorocarbobenzyloxy, carbobenzyloxy, 6. A resin peptide having the structure: Lbromocarbobenwlow,

dichlorocarbobenzyloxy, or trifluoroacetyl. NH,-Lys-Val-Tyr-Pro-Asn-Gly-Ala-GlLi-As T-Glii-SeT-Ala-GlT-Ala- A resin Peptide having the Structure:

V Y B2 B2 B1 B2 B1 r MH -Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lyis-Pro-val-Gly- Hz Y 132 B2 W Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asp-Ala-Gly-Glu-As in which I l l l T 82 is benzyl, p-methoxybenzyl, p-chlorobenzyl, pv T T v B2 B2 B2 nitrobenzyl or benzylhydryl, 40

Gln-Se r-AlaGlu-Ala-Phe-Pro-Leu-Glu-Phe-CH,-

Y is B2 or H, B B B V is 2-chlorocarbobenzyloxy, carbobenzyloxy, 2- Z z z bromocarbobenzyloxy, 2,4-

. in which dichlorocarbobenzyloxy, or trifluoracetyl B2 is benzyl, p-methoxybenzyl, p-chlorobenzyl, p-

and is divinylbenzene crosslmked polystyrene resin nitrobenzyl or benzylhydryl,

7 A resin peptide having the Structure is divinylbenzene crosslinked polystyrene resin,

Y is 82 or H, V is 2-chlorocarbobenzyloxy, carbobenzyloxy, 2- NH Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Glybromocarbobenzyloxy, 2,4-

81 Y B2 82 w I I dichlorocarbobenzyloxy, or trifluoroacetyl T is tosyl or nitro I f' T Q and W is carbobenzyloxy, tosyl, dinitrophenyl, B2 or v T 82 B2 11. In a process for synthesizing a resin peptide, the step of coupling B2 B2 B2 B2 2- with the reactant GIu-Ser Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe-CH in which B2 is benzyl, p-methoxybenzyl, p-chlorobenzyl, p-

nitrobenzyl or benzylhydryl, P Glu Y is 82 or H, Bz V is 2-chlorocarbobenzyloxy, carbobenzyloxy, 2-

bromocarbobenzyloxy, 2,4- or an active ester or an azide thereof, said coupling dichlorocarbobenzyloxy, 01' trifluoroacetyl, being conducted in the presence of a diimide unless T is tosyl or nitro, said reactant is an active ester or an azide, to produce W is carbobenzyloxy, tosyl, dinitrophenyl, B2 or H the peptide where P is tertiary-butyloxycarbonyl, amyloxycarbonyl, or

o-nitrophenylsulfenyl,

Bz is benzyl, p-methoxybenzyl, p-chlorobenzyl, p-

nitrobenzyl or benzylhydryl and (B) is divinylbenzene crosslinked polystyrene resin.

12. The process of claim 11 including the additional step of contacting the reaction product of claim 1 l with an acid to remove P therefrom.

13. The process of claim 11 including the additional step of coupling with the reactant P-l-leucine or an active ester or an azide thereof, said coupling being conducted in the presence of a diimide unless said reactant is an active ester or an azide, to produce Y Bz BzW Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu- V T V Y B2 B2 B2 Ser-Ala-GlwAla-Phe-Pro-Leu-Glu-Phe-CH with the reactant P-Bz-l-serine or an active ester or azide thereof, said coupling being conducted in the presence of a diimide unless said reactant is an active ester or an azide, to produce NH -Ser-Tyr-Ser-Met-Glu-His-Phe-Aijg-Trp-Gly-Lyis- BZY Bz BZW Pro-Val-Gly-Lys-Lys-Arg-A1g-Pro-Val-uis-ValTTr-Pro-Asn-Gly VVT Y Ala-Glu Asp-Glu-Ser-Ala-Glu-Ala-PhePro-Leu-Glu-Phe-CH where B2 is benzyl, p-methoxybenzyl, p-chlorobenzyl, p-

nitrobenzyl or benzylhydryl,

is divinylbenzene crosslinked polystyrene resin,

Y is B2 or H,

V is 2-chlorocarbobenzyloxy, carbobenzyloxy, 2- bromocarbobenzyloxy, 2,4- dichlorocarbobenzyloxy, or trifluoracetyl,

T is tosyl or nitro,

W is carbobenzyloxy, tosyl, dinitrophenyl, B2 or H and P is tertiary-butyloxycarbonyl, amyloxycarbonyl,

or o-nitrophenylsulfenyl.

16. The process of claim 15 including the additional step of contacting the reaction product of claim 15 with anhydrous hydrogen fluoride to remove the--CH 82, T, Y and W groups.

17. The process of claim 16 in which V is trifluoracetyl and including the additional step of dissolving the reaction product of claim 16 in an aqueous base to remove the trifluoroacetyl protecting groups.

18. In a process for synthesizing a resin peptide, the step of coupling NH -Tyr-Ser-Met-GlT-HiT-Phe-A1ig-Trp-Gly-Lyis-Pro-vabGly-Lyis- LysArg-Arg- Pro-Val-Lys-ValTyr-Pro-Aj-Ala-Gly-GluAsT-Gln- V 2 B2 B2 Ser-Ala-Glu-Ala-Phe-PrmLeu-Glu Phe-CH with the reactant P-Bz-l-serine or an active ester or an azide thereof, said coupling being conducted in the presence of a diimide unless said reactant is an active ester or an azide, to produce NH,-Ser-Tyr-Ser-Met-Glu-His'lhe-Arg-Trp-Gly-Lys-Pro-Val- Gly-Lys-Lys-Arg-Alg-Pro-Val-Lyis-val-TTr-Pro-AsiAla-Gly- VV Y Bz acetyl and including the additional step of contacting the reaction product of claim 19 with an aqueous base to remove the trifluoroacetyl protecting groups.

21. A resin peptide as set forth in claim 3 in which 82 is benzyl.

22. A resin peptide as set forth in claim 4 in which 82 is benzyl.

23. A resin peptide as set forth in claim 5 in which 82 is benzyl.

24. A resin peptide as set forth in claim 7 in which T is tosyl.

25. A resin peptide as set forth in claim 7 in which V is trifluoroacetyl and B2 is benzyl.

26. A resin peptide as set forth in claim 8 in which B2 is benzyl.

27. A resin peptide as set forth in claim 9 in which V is trifluoroacetyl.

28. A resin peptide as set forth in claim 10 in which V is trifluoracetyl.

29. A resin peptide as set forth in claim 10 in which T is tosyl.

30. A resin peptide as set forth in claim 10 in which V is trifluoracetyl and B2 is benzyl.

31. A process as set forth in claim 11 in which B2 is benzyl.

32. A process as set forth in claim 13 in which BZ is benzyl.

33. A process as set forth in claim 18 in which Hz is benzyl. I i

34. A process as set forth in claim 18 in which V is trifluoracetyl.

35. A process as set forth in claim 18 in which T is tosyl.

36. A process as set forth in claim diimide is dicyclohexyl carbodiimide.

37. A process as set forth in claim diimide is dicyclohexyl carbodiimide.

38. A process as set forth in claim diimide is dicyclohexyl carbodiimide.

39. A process as set forth in claim diimide is dicyclohexyl carbodiimide.

11 in which said 13 in which said 15 in which said 18 in which said Patent 3 915 949 Dated October 28, 1975 Column 3 Column 4, line 35, "bya" should read by a read E 2 should read 2 both occurrences.

. Column 5, line 11 "reaction of the P, B2 and glutamic" should UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Robert L. Colescott et al Page 1 of 3 Inventor-(s) It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 2, line 22, "Cl-CH' OCH" should read C1CH OCH Column 2, line 25, "divinly benzene" should read divinylbenzene line 1 "PHENYLALAMINE" should read PHENYLALANINE I Column 3, lines 28 and 29, "tertbutyloxy-lphenylalamine" should read tert-butyloxy phenylalanine same line 35, after "125" insert ml Column 4, lines 35 and 36, delete "by a".

Column 4, lines 41 and 51, for that portion of the formula read reaction, the P, B2 and glutamic Column 5, line 16, "phenylalanie" should read phenylalanine Column 6, line 40, 45 and 52, "BOC-l-r-benzyl", each occurrence,

should read BOC-l- X-benzyl UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Q pa 4 Dated October 28, 1975 Robert L. Colescott et al Page 2 of 3 Inventor(s) It is certified that error appears in the aboveidentified patent and that said Letters Patent are hereby corrected as shown below:

Column 7, line 23, "BOC-lrbenzylglutamate" should read BOC l-K-benzyl glutamate Column 8, line 37, "that" should read at Column 9, line 36, "carbobenzyl (CBZ)," should read carbobenzyloxy (CBZ) Column 9, line 65, "positions" should read position Column 10, line 15, CH Phe-" should read -CH -Pheb Column 10 line 50, "Ala-G1y-Asn-Pro-Tyr" should read Column 10 line 55 "this" should read the Column 11, line 7, "Phe-Ais-Glu-" should read Phe-His-Glu- WBz WBZ b Column 13, line 34, "BOC-lr benzyl' should read BOG-1:5

benzyl UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Pa 4 Dated October 28, 1975 Robert L. Colescott et a1 Page 3 f 3 Inventor (s) It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 14, line 9, "Boc-1-P-benz 1" should read BOC-l-X benzyl Column 15, lines 42 to 45, delete "may be purified as described in connection with compound 40 and".

Column 15, line 55, "ia" should read is Column 16, line 51, delete "and" and line 52 the comma, should read and Claim 17, lines 1 and 2, "trifluoracetyl" should read trifluoracetyl Signed and Scaled this Eighteenth D3) of January 1977 [SEAL] A ttes t:

RUTH C. MASON C. MARSHALL DANN Arresting Officer Commissioner oj'Patems and Trademarks

Non-Patent Citations
Reference
1 *Basjusz et al., Chem. Abstr., 71:70936z, (1969)
2 *Basjusz et al., Chem. Abstr., 71:77857h, (1969)
3 *Merrifield, Adv. in Enzymology, 32, 244-247, (1969)
4 *Yajima et al., Chem. Pharm. Bull., 18, 1333-1339, (1970)
5 *Yamashiro et al., J. Am. Chem. Soc., 95, 1310-1315, (1973)
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US4130514 *Feb 10, 1978Dec 19, 1978Armour Pharmaceutical CompanySynthesis of peptides
US4242238 *Feb 13, 1976Dec 30, 1980Armour And CompanyOligo- and resin peptides, acth(1-28)
US4301045 *May 2, 1977Nov 17, 1981Armour Pharmaceutical CompanyResin peptides
US4327075 *Apr 14, 1980Apr 27, 1982Ab BonnierforetagenImmunology
US4336187 *Sep 22, 1980Jun 22, 1982Armour Pharmaceutical CompanyButanol and lower alkanol as solvents acetic acid, ammonium acetate
US4487715 *Jul 9, 1982Dec 11, 1984The Regents Of The University Of CaliforniaMethod of conjugating oligopeptides
US4794104 *Jan 2, 1987Dec 27, 1988Alfio BertoliniInjection
Classifications
U.S. Classification530/334, 930/280, 930/70, 530/306, 514/805, 930/DIG.571
International ClassificationC07K14/575, A61K38/00, C08F8/30, C07K14/695, A61P5/38, C07K1/04, C08H1/00, C08F8/00, C08F8/24, C07K17/08
Cooperative ClassificationA61K38/00, C07K1/042, C08H1/00, C08F8/30, Y10S930/28, C07K14/695, Y10S514/805
European ClassificationC08F8/30, C07K1/04A, C07K14/695, C08H1/00